This invention relates to the biochemistry of cyclophilin proteins, in particular, compounds which interact with or bind such proteins. Cyclophilins (CyP), which bind cyclosporin A, and FK-506 binding proteins (FKBP), which bind FK-506 and rapamycin, are both subclasses of a group of proteins termed immunophilins. Immunophilins were first identified as proteins that bind to the immunosuppressive drugs cyclosporin A, FK-506, and rapamycin. CyPs and FKBPs can also be separated based on their differing structures.
By studying the binding of test compounds to cyclophilin proteins, the inventors have identified a number of new compounds that effect the growth and health of cells in the nervous system. Building on this initial identification, the inventors developed and utilized screening procedures for rapidly identifying additional, similarly active compounds. These compounds have been specifically tested to show that they protect neuronal cells from otherwise lethal treatments, and/or that they promote the growth or regeneration of neuronal cells. In part, the invention provides compounds that interact with or bind to a cyclophilin and compounds that have activity towards neuronal cells. The compounds can be used in a variety of ways, including therapeutic and research and development applications for a number of diseases associated with neuronal degeneration.
Cyclophilin was first identified as the receptor for cyclosporin A, a potent immunosuppressive drug that is still widely used to prevent immunological rejection of transplanted tissue. The effects of the cyclosporin A:cyclophilin interaction have been well documented. Cyclosporin A binds with a dissociation constant in the range of 10xe2x88x928 mol/L, a value representing a relatively high degree of attraction (Handschumacher et al., Science 226:544 (1984)). While the present invention is not bound by any particular theory, it appears the complex formed between CyP and cyclosporin A exerts the effects on the organism and cells, which leads to immunosuppression. The complex interacts with the cellular enzyme calcineurin, a calmodulin-dependent phosphatase, and the interaction prevents T cell activation by blocking RNA transcription of the T cell growth factor interleukin 2 (IL-2). (Palacios, J. Immunol. 128:337 (1982)). Without IL-2 to cause T cell proliferation, specific T cell populations cannot mount a strong immune response, resulting in immunosuppression.
A number of types of mammalian cyclophilins have been identified and cloned, cyclophilins A, B, C, D, and cyclophilin-40 (Snyder and Sabatini, Nat. Med. 1:32-37 (1995); Friedman et al., Proc. Natl. Acad. Sci., 90:6815-6819 (1993)). Cyclophilin A is a 19 kD protein, which is abundantly expressed in a wide variety of cells. Like the other cyclophilins, cyclophilin A binds the immunosuppressive agent cyclosporin A and possesses peptidyl-prolyl cis-trans isomerase (PPIase) and protein folding or xe2x80x9cchaperonexe2x80x9d activities. PPIase activity catalyzes the conversion of proline residues in a protein from the cis to the trans conformation (Fischer, et al., Biomed. Biochem. Acta 43:1101-1112 (1984)). Cyclophilin B possesses an N-terminal signal sequence that directs translocation into the endoplasmic reticulum of the cell. The 23 kD cyclophilin C is found in the cytosol of the cell. Cyclophilin D, at 18 kD, appears to target its actions in the mitochondria. And cyclophilin-40 is a component of the inactivated form of a glucocorticoid receptor.
Immunophilins were discovered because of their interaction with known therapeutic drugs. Thus, knowledge about the interaction between drug and protein spawned a number of drug discovery efforts. Initially, the focus was on identifying new immunosuppressive drugs. A number of facts have influenced the search for improved immunosuppressive drugs. One factor was the importance of proline. The native substrate for the PPIase activity in cells is the amino acid proline in a protein. Cyclophilins A-D all contain a conserved proline binding site. The conversion between the cis and trans forms of proline, which PPIase performs, allows a protein to change shape and fold properly.
However, the first identified ligand for cyclophilins, cyclosporin A, which is a cyclic peptide, does not contain a proline. Both FK-506 and rapamycin, which bind FKBP, are also cyclic non-peptidic macrolide antibiotics. The FKBP proteins also possess PPIase activity, although the FKBPs share no significant sequence homology to CyPs. Since FK-506 is a more potent immunosuppressive compound than cyclosporin A, a number of analogs of FK-506 have been developed. So, the cyclic structure also became an important factor in designing potential new drugs.
Later, therapeutic applications in the nervous system were identified (Lyons et al., PNAS 91:3191-3195 (1994)). A number of animal models have proven the effectiveness of FKBP ligands in promoting nerve regeneration and nerve growth. (See, for example, Steiner et al., PNAS 94:2019-2024 (1997); Hamilton et al., Bioorg. Med. Chem. Lett. 7:1785-1790 (1997); Gold et al., Experiment. Neurol. 147:269-278 (1997); and Wang et al., J. Pharm. Exp. Therap. 282: 1084-1093 (1997).) However, whether or not ligands specific for CyP possess similar activity in the nervous system has been controversial (Hamilton and Steiner, J. Med. Chem. 41:5119-5143 (1998); Gold, Mol. Neurobiol. 15:285-306 (1997); and Carreau et al., Neuropharmacol. 36:1755-62 (1997)). Earlier published work by some of the inventors showed how compounds with an affinity for the cyclophilin immunophilins can be useful in effecting neuronal activity (PCT published applications WO 97/18828 and WO 98/25950). The work of the present invention further demonstrates that ligands specific for CyP are active in the nervous system and expands on the earlier work by providing additional structural and functional aspects.
Researchers have also noted a functional association of cyclophilin A with the Gag protein of the HIV virus (Thali et al., Nature 372:363-365(1994)). This has taken drug development approaches in a new direction (See, for example, U.S. Pat. No. 5,767,069). Many researchers now seek to develop drugs that target the interaction between cyclophilin A and Gag in order to disrupt the HIV life cycle (Sternberg, BioWorld Today 7:1 (1996)).
The invention provides a number of compounds that bind to CyP proteins as well as compounds that are structurally or functionally related to those specifically described and shown. The compounds of this invention preferably do not suppress the immune system and preferably do not possess a biological activity involving binding to a FKBP, i.e., the compounds have an IC50 greater than 10 xcexcM towards FKBP. A number of methods for determining the binding to CyPs are presented and so are a number of ways for exploiting the binding through in vitro and in vivo methods and uses. Preferred compounds function to promote or affect neuronal cell growth or growth of nervous system cells, regenerate damaged or diseased neurons, or protect neurons or neuronal cells from damage. Furthermore, aspects of this disclosure can be used in methods to identify and isolate additional CyP binding compounds or additional uses of the compounds.
The invention also provides a number of uses for these compounds, including uses that comprise the step of allowing the compound to contact an immunophilin protein. A variety of permutations of this method can be devised. In particular, the compounds can be used to affect neuronal cells, either in culture or in an animal. Thus, the compounds can be administered to cells or animals to affect a number of conditions associated with the decline, damage, or degeneration of nervous system cells or function.
In one aspect, this invention provides compounds of Formula I and Formula II, shown and described below. 
where n in Cn is 0 or 1;
the dashed bond symbol represents an optional bond;
X and Y may independently be N, NH, O, S, or a direct bond;
R1 is the same or different from R2, and either can be
one or more C1-C6 branched or straight chain alkyl or alkenyl groups;
one or more C1-C3 branched or straight chain alkyl groups substituted by one or more Q groups;
or one or more Q groups,
where Q, which is optionally saturated, partially saturated, or aromatic, is a mono-, bi-, or tricyclic, carbo- or heterocyclic ring, wherein each ring may be optionally substituted in one to three positions with halo, hydroxyl, nitro, trifluoromethyl, acetyl, aminocarbonyl, methylsulfonyl, oxo, cyano, carboxy, C1-C6 straight or branched chain alkyl or alkenyl, C1-C4 alkoxy, C1-C4 alkenyloxy, phenoxy, benzyloxy, amino, or a combination thereof, and wherein the individual ring sizes are 5-6 members, and wherein each heterocyclic ring contains 1-6 heteroatoms selected from the group consisting of O, N, S, or a combination thereof;
and R3 many be one to three substituents chosen from the group consisting of halo, hydroxyl, nitro, trifluoromethyl, C1-C6 straight or branched chain alkyl or alkenyl, C1-C4 alkoxy, C1-C4 alkenyloxy, phenoxy, benzyloxy, amino, Q as defined above, or a combination thereof. 
where R4 and R5 may independently be
xe2x80x94Nxe2x80x94SO2xe2x80x94R,
xe2x80x94SO2xe2x80x94NRR,
xe2x80x94Oxe2x80x94R,
xe2x80x94COxe2x80x94Nxe2x80x94R,
xe2x80x94Nxe2x80x94COxe2x80x94R,
xe2x80x94COxe2x80x94R,
wherein each R may independently be
hydrogen, Q, or a C1-C6 branched or straight alkyl or alkenyl chain, which may be substituted in one or more positions by C3-C8 cycloalkyl or cycloalkenyl, hydroxyl, or carbonyl oxygen, and where in said alkyl or alkenyl chain one or more carbon atoms are either optionally substituted with Q, or optionally replaced by O, S, SO, SO2, N, or NH;
where Q, which is optionally saturated, partially saturated, or aromatic, is a mono-, bi-, or tricyclic, carbo- or heterocyclic ring, wherein each ring may be optionally substituted in one to five positions with halo, hydroxyl, nitro, trifluoromethyl, acetyl, aminocarbonyl, methylsulfonyl, oxo, cyano, carboxy, C1-C6 straight or branched chain alkyl or alkenyl, C1-C4 alkoxy, C1-C4 alkenyloxy, phenoxy, benzyloxy, amino, or a combination thereof, and wherein the individual ring sizes are 5-6 members, and wherein each heterocyclic ring contains 1-6 heteroatoms selected from the group consisting of O, N, S, or a combination thereof.
In a preferred embodiment of a compound of Formula II, each R in R4 and R5 may independently be
hydrogen, Q, or C1-C6 branched or straight chain alkyl or alkenyl, which may be substituted in one or more positions by C3-C8 cycloalkyl or cycloalkenyl, hydroxyl, carbonyl oxygen, or Q;
where Q, which is optionally aromatic, is a mono-, bi-, or tricyclic, carbo- or heterocyclic ring, wherein each ring may be optionally substituted in one to three positions with halo, hydroxyl, nitro, trifluoromethyl, C1-C6 straight or branched chain alkyl or alkenyl, C1-C4 alkoxy, C1-C4 alkenyloxy, phenoxy, benzyloxy, amino, or a combination thereof, and wherein the individual ring sizes are 5-6 members, and wherein each heterocyclic ring contains 1-6 heteroatoms selected from the group consisting of O, N, S, or a combination thereof.
A number of compounds can be selected for use from Formulae I and II. For example, starting with a particular compound, any of the individual variable groups R1-R5, X, Y, and a value for xe2x80x98nxe2x80x99 can be selected while one or more of the other variable groups can be modified. For example, in Formula I, the xe2x80x9cnxe2x80x9d can be set at 0 to select subgroups of related compounds with X and Y being both NH, or both being O, or X being NH and Y being O, and within each of those 3 groups R3 being present or absent, and then within each of those 6 groups the 6-membered ring structure is either a cyclohexyl or an aromatic ring, which results in 12 subgroups of related compounds. Any of those 12 subgroups can be selected and further divided into additional subgroups of compounds defined by having an R1 the same as R2 or by having both R1 and R2 comprise a substituted benzyl or substituted phenyl group. This process can be repeated using any one or combination of the variable groups. In this way, one skilled in the art can select and use groups of related compounds or even individual compounds, all within the invention. Many examples are shown below; however, they are merely representative of the scope of changes and modifications possible. One skilled in the art can devise many separate compounds from the description of the Formulae alone. Thus, the invention specifically includes numerous individual compounds that fall within the definition of either Formula I or II.
Compounds of Formulae I and II may be prepared or formulated as a salt or derivative for some uses, including pharmaceutical and tissue or cell culture uses. The compounds of the invention can also be part of a composition comprising one or more compounds of Formula I or II. Thus, pharmaceutically acceptable salts and derivatives of any of the compounds, or compositions comprising them, are specifically included in this invention. A compound of Formula I or II, or a compound having Formulae I or II, will optionally include the salt or derivative of the compound depicted in the formula.
The compounds of the invention can be produced as a mixture of isomers or racemic mixtures or as optically pure compounds. Methods for separating stereoisomers can also be used to enrich mixtures for one or more compounds. The compositions of the invention may similarly contain mixtures of stereoisomers, mixtures of one or more stereoisomers, or be enriched for one or more stereoisomers. All of these forms are specifically included in this invention.
Preferably, compounds of Formulae I and II selectively bind to a CyP as detected, for example, by a measurable inhibition of the rotamase (PPIase or peptidyl-prolyl cis-trans isomerase enzyme) activity of CyP. xe2x80x9cSelectively bind to a CyPxe2x80x9d means the compounds do not possess a significant binding affinity toward a FKBP and/or do not possess a biological activity associated with binding to a FKBP. For example, the IC50 towards FKBP is at or above 10 xcexcM or at or above 50 xcexcM. The skilled artisan is familiar with ways to detect rotamase inhibition in CyP and FKBP. In addition, a number of ways for detecting binding to a CyP are described below.
As is readily apparent from Formulae I and II, a common 1-,3-substitution pattern on a central ring structure exists. This common pattern differs from the approaches previously taken to identify other immunophilin binding compounds or drugs. For example, Holt et al. (Bioorg. Med. Chem. Letters, 4: 315-320 (1994)) discuss a pipecolate, or 1-(1,2-dioxo)2-carboxylate piperidine containing base structure for binding to FKBP. Similarly, earlier work by the inventors established the relevance of a 1-(1,2-dioxo)2-carboxylate pyrrolidine containing structure for binding to FKBP (Steiner et al., PNAS 94:2019-2024 (1997)). Presumably, these structures mimic the natural substrate for the rotamase activity, a proline-containing fragment of a protein. In a protein, the amino acid proline corresponds to a 1,2-substituted pyrrolidine structure. Prior work has generally incorporated that structure. However, Formulae I and II do not correspond to a 1,2- substituted pyrrolidine structure. Yet, as demonstrated here, compounds of these formulae possess important bioactive and biochemical functions.
The body of work related to analogues of cyclosporin A, FK-506, and rapamycin further distances the compounds of this invention from prior work. (See, for example, U.S. Pat. Nos. 5,767,069, 5,284,826, 4,703,033, and 5,122,511.) These analogues typically possess a cyclic peptide structure.
In another aspect, the invention relates to methods for binding non-peptidic compounds to cyclophilin-type immunophilins. Binding results in an xe2x80x9cimmunophilin:drugxe2x80x9d complex, which is considered to be the active agent in the in vivo immunosuppressive and neurotrophic activities of rotamase inhibitors (Hamilton and Steiner, J. of Med. Chem. 41:5119-5143 (1998); Gold, Mol. Neurobiol. 15:285-306 (1997)). Whether or not the complex acts for any or all the therapeutic actions of these rotamase inhibitors, focusing on the immunophilin:drug interaction has led to the discovery a number of new drug compounds. Accordingly, methods of using compounds, such as those of Formulae I and II, to create an immunophilin:compound complex, or a CyP:compound complex, provides an important aspect of this invention. This aspect can be exploited, for example, in methods where the compound, or a mixture comprising one or more of the compounds of the invention, is administered to cells in culture or to an animal.
While the immunophilin:compound complex has beneficial effects in vivo and in cultured cells, numerous other uses for binding the compounds to an immunophilin exist. For example, in vitro binding experiments can be used to identify and purify cellular components that interact with the immunophilin complex. An affinity chromatography column or matrix bearing the compound can be reacted with a CyP, and cellular or tissue extracts passed over the column or matrix.
Thus, the invention also provides methods for forming immunophilin:compound or CyP:compound complexes as well as the complexes themselves. To form these complexes, the compounds can contact an immunophilin or CyP protein in vivo, in vitro, or within a cell. In preferred embodiments, the compound contacts a human CyP protein, such as one or more of CyP A, B, C, or D. The CyP protein can be native to the cell or organism, produced via recombinant DNA, produced by other manipulations involving introduced genetic material, or produced by synthetic means. Furthermore, chimeric proteins possessing immunophilin domains that function to bind immunophilin ligands can also be used to form a protein:compound complex. The formation of the CyP:compound, immunophilin:compound, or protein:compound complex need not be irreversible.
The binding of a compound to a CyP can be detected in a number of ways, including rotamase inhibition assay, affinity chromatography, in vivo neuroprotection or neuroregeneration activity assay, in vitro neurotrophic activity assay, or by any of the activities in neuronal cells or cells of the nervous system described below, in the examples, or in the cited references.
The invention also provides compositions comprising at least one compound of Formula I or II. The compositions may comprise one or more pharmaceutically acceptable carriers, excipients, or diluents. These compositions, or the compounds themselves or mixtures of them, can be administered to an animal. Administration can be one method to allow the compound to contact a CyP within the animal. As one skilled in the art would recognize, various routes of administration are possible. Exemplary routes are specifically described in the detailed description below. The compounds of Formulae I and II or compositions comprising them can function to regenerate nerve cells, promote neurite outgrowth, and protect nerves from otherwise damaging treatments or conditions. Thus, the compounds and compositions of this invention can be used to treat animals, including humans, with neurodegenerative conditions or animals exposed to degenerative agents or having damaged nervous system cells.
The following detailed description should not be taken as a limitation on the scope of the invention. The embodiments and examples given are illustrative of the invention. Additional aspects of the invention can be devised by reference to this disclosure as a whole in combination with the references cited and listed throughout and at the end of the specification and the knowledge of one skilled in the art. All of the references cited and listed can be relied on, in their entirety, to allow one to make and use these additional aspects of the invention.